Methods for isolating a target nucleic acid from red blood cells

ABSTRACT

The invention provides a lysis reagent for lysing red blood cells, thereby releasing a target, such as RNA from a parasitic organism, in a form suitable for analysis. The reagent includes at least ammonium chloride and an anionic detergent, and may include an anti-coagulant. The reagent serves to lyse red blood cells, protect the released target from degradation in the lysate, and is compatible with subsequent steps for analysis of the target such as target capture, amplification, detection, or sequencing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/019,829, filed Jun. 27, 2018, now issued as U.S. Pat. No. 10,689,713,which is a division of U.S. patent application Ser. No. 14/918,131,filed Oct. 20, 2015, now issued as U.S. Pat. No. 10,093,989, whichclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 62/066,244, filed Oct. 20, 2014, each of which isincorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on May 6, 2020, isnamed “GP323-02UT_Seq_List_ST25” and is 3,030 bytes in size.

BACKGROUND

Although there are commercial assays for detecting RNA in blood, the RNAdetected in such assays is usually present in extracellular forms, suchas HIV or HCV particles in the blood. Detection of RNA or other targetmolecules from within red blood cells is more challenging. Reagents usedin lysis may interfere with subsequent processing as many non-targetmolecules released by lysis, particularly nucleases or proteases, maydegrade target molecules.

The intrinsic instability of RNA and presence of RNAses in whole bloodmakes isolation of RNA a difficult task. The use of high purity, intactRNA facilitates sensitive clinical diagnostic assays. Existingapproaches typically involve several sequential steps: a step to disruptthe cells, a step to denature the proteins, another step for thestabilization and protection of RNA from RNAses, and then a step forisolation of the RNA

Tetradecyltrimethylammonium oxalate (TDTMAO) is commonly used fortransport, storage and processing of blood (U.S. Pat. Nos. 6,602,718 and6,617,170). This quaternary amine is contained, for example, in thePAXgene™ Blood RNA System (BD Biosciences) and works by penetrating thecell and stabilizing intracellular target RNA. The RNA can then be laterpurified and analyzed from the components of whole blood using standardtechniques. Methods for lysing cells and inhibiting RNases usingguanidinium salts are also known (Chomczynski et al. (1987) Anal.Biochem. 162, 156-159).

Human Babesiosis is an emerging infectious disease resulting from atick-borne intraerythrocytic infection of red blood cells. Babesiamicroti, the most common cause for Babesiosis in the United States iswide epidemic in the Northeastern and upper Midwestern states. Thisspecies has been implicated in a majority of the transfusion-transmittedBabesiosis (TTB) cases in the United States and is currently the mostreported transfusion-transmitted disease to the FDA. Between 2005 and2010, 3.6% of transfusion-related deaths reported to the FDA wereattributed to TTB. However, there is no FDA approved test for bloodscreening for Babesia to date despite the risk that this agent poses tothe United States blood supply.

SUMMARY OF THE CLAIMED INVENTION

The invention provides a reagent comprising ammonium chloride, lithiumlauryl sulfate (LLS), and an anti-coagulant. In some embodiments, thereagent further comprises a buffer. In some embodiments, the buffer issodium bicarbonate. In some embodiments, the pH of the buffer is 7-8 or7.2-7.6. In some embodiments, the anti-coagulant is EDTA, heparin, orcitrate.

In some embodiments, the concentration of ammonium chloride in thereagent is 100-500 mM, 200-300 mM, or 250 mM. In some embodiments, theconcentration of LLS in the reagent is 4-15% (w/v), 5-8% (w/v), or 5%(w/v). In some embodiments, the concentration of sodium bicarbonate inthe reagent is 5-30 mM, 10-20 mM, or 14 mM. In some embodiments, theconcentration of ammonium chloride is 250 mM, the concentration of LLSis 5% (w/v), the concentration of sodium bicarbonate is 14 mM, and thepH is 7.2-7.6.

In some embodiments, the reagent is admixed with red blood cells orproducts derived from red blood cells. In certain embodiments, thereagent is admixed with whole blood. In some embodiments, the reagent isadmixed with whole blood in a ratio of 3:1 (v/v). In some embodiments,the whole blood is human whole blood, non-human whole blood, or amixture thereof.

The invention further provides a method of analyzing a target from redblood cells comprising: (a) contacting red blood cells with a reagentcomprising ammonium chloride and an anionic detergent, the reagent beingeffective to lyse the red blood cells and inhibit degradation of targetreleased from the red blood cells; and (b) analyzing the target releasedfrom the red blood cells.

In some methods, the target is a pathogen-derived target. In somemethods, the target is RNA.

In some methods, at least 50% of the red blood cells are lysed in fiveminutes or less. In some methods, the percentage of lysed red bloodcells is higher than the percentage of lysed white blood cells.

In some methods, analyzing the target comprises contacting the releasedtarget with a capture probe and an immobilized probe, the capture probehaving a first segment complementary to the target, and a second segmentcomplementary to the immobilized probe, wherein the target binds to thecapture probe, and wherein the bound capture probe binds to theimmobilized probe. Some methods further comprise performing atranscription mediated amplification of the target and detecting theresulting amplification product with a detection probe.

Some methods are performed without a centrifugation step to separate thereagent from the target released from the red blood cells.

In some methods, the target is 18S rRNA from a pathogenic organism ofthe genus Babesia. In some methods, the pathogenic organism is of thespecies Babesia microti. In some methods, the limit of detection is atleast Babesia 2×10³ copies of Babesia microti 18S rRNA per 1 mL of wholeblood.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows detection of Babesia 18S rRNA in human whole blood spikedwith Babesia-infected hamster blood. Samples were lysed with a lysisreagent of buffered ammonium chloride (ACL) at a concentration of 250mM. Babesia 18S rRNA was detected using PROCLEIX® target capture reagent(TCR) and transcription mediated amplification (TMA).

FIGS. 2a, 2b and 2c show analysis of various lysis reagents and theirability to lyse whole blood samples without causing cell precipitation,cell clumping, cell debris, or magnetic bead loss when added to TCR foranalysis.

FIG. 3 shows the compositions of various lysis reagents and theirpotential use for lysing whole blood samples for detection of Babesia18S rRNA.

FIG. 4 shows a comparison between PTM2.4 (250 mM ACL, 14 mM sodiumbicarbonate, 5% lithium lauryl sulfate (LLS)) and IC buffer (10% LLS)for use in lysing whole blood samples prior to detection of Babesia 18SrRNA.

FIG. 5 shows the effect of LLS in the buffered ACL solution fordetection of Babesia 18S rRNA in whole blood samples.

FIG. 6A shows assay sensitivity for detecting Babesia 18S rRNA in humanwhole blood samples spiked with serial dilutions of Babesia-infectedhamster blood. Samples were lysed with a lysis reagent of buffered ACLand 5% LLS prior to detection.). Arbitrary cutoff=50,000 RLU; Anyactivity >50,000 RLU is considered 100% reactive.

FIG. 6B shows the sensitivity of an assay for detecting Babesia 18S rRNAin vitro transcripts spiked into human whole blood. Spiked samples werelysed with a lysis reagent of buffered ACL and 5% LLS prior todetection.). Arbitrary cutoff=50,000 RLU; Any activity >50,000 RLU isconsidered 100% reactive.

FIG. 7 shows a comparison between two ratios of lysis reagent to humanwhole blood for the detection of Babesia 18S rRNA. A lysis reagent ofbuffered ACL and 5% LLS was used for cell lysis. The ratios of humanwhole blood to lysis reagent evaluated in the experiment were 1:2 and1:3 (blood:reagent). Arbitrary cutoff=50,000 RLU; Any activity >50,000RLU is considered 100% reactive.

FIG. 8 shows reproducibility of an assay for detecting Babesia 18S rRNAin human whole blood samples obtained from 6 different donors. Arbitrarycutoff=50,000 RLU; Any activity >50,000 RLU is considered 100% reactive.

FIG. 9 shows stability of Babesia parasites in human whole blood. Serialdilutions of Babesia-infected hamster blood were spiked into human wholeblood. One group was stored for 5 days at 25° C. prior to analysis.Another group was analyzed immediately after addition of theparasite-infected blood. The infected whole blood samples were lysedwith a lysis reagent of buffered ACL and 5% LLS prior to detection ofBabesia 18S rRNA. Arbitrary cutoff=50,000 RLU; Any activity >50,000 RLUis considered 100% reactive.

FIG. 10 shows the stability of Babesia 18S rRNA released from red bloodcells. Serial dilutions of Babesia-infected hamster blood were spikedinto human whole blood. Samples were lysed with a lysis reagent ofbuffered ACL and 5% LLS. Following lysis, samples were stored at 4° C.for 0, 1, 3, or 4 days prior to analysis for Babesia 18S rRNA. Arbitrarycutoff=50,000 RLU; Any activity >50,000 RLU is considered 100% reactive.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 sets forth the nucleic acid sequence of a non T7 primer.

SEQ ID NO:2 sets forth the nucleic acid sequence of a T7 primer.

SEQ ID NO:3 sets forth the nucleic acid sequence of an acridinium ester(AE) probe.

SEQ ID NO:4 sets forth the nucleic acid sequence of a target captureoligonucleotide (TCO) probe.

SEQ ID NO:5 sets forth the nucleic acid sequence of an in vitrotranscript copy of Babesia 18S rRNA.

Definitions

Pathogens include viruses, bacteria, protozoa, fungi, and othermicroorganisms responsible for disease in humans and other animals.

A target can be a single type of molecule, such as a protein or 18S rRNAof Babesia, or a class of molecules, such as any protein or RNA fromBabesia or any protein or RNA from red blood cells. Multiple distincttargets can also be analyzed, such as an RNA target and a proteintarget, or two distinct RNA targets, such as two different mRNA targets,or an mRNA target and an rRNA target. Targets include endogenouscomponents of red blood cells and components arising as a result ofpathogenic infection of infected red blood cells and are typicallyencoded by the infecting pathogen (i.e., “pathogenic” or“pathogen-derived” targets).

A lysis reagent is reagent, often provided in the form of a solution,effective for inducing lysis of red blood cells in whole blood or redblood cell products such as pelleted red blood cells.

Preferential lysis of red blood cells over other cellular components ofblood means that the percentage of red blood cells lysed is higher thanthat of other cellular components present in the sample being analyzed,the other cell types being assessed in the aggregate.

Anionic detergents are compounds with a negatively charged, anionic headgroup and a long hydrocarbon tail, often provided as a salt with analkali metal or ammonium ion.

Anti-coagulants inhibit clotting of whole blood. Anti-coagulants includeheparins and calcium chelating agents. Heparins activate antithrombinIII, which inhibits the activity of thrombin and other proteasesinvolved in blood clotting. Calcium chelating agents, such as EDTA andcitrate, bind calcium ions required for blood clotting.

A buffer refers to a weak acid or weak base used to maintain the pH of asolution.

A nucleic acid refers to a multimeric compound comprising nucleotides oranalogs that have nitrogenous heterocyclic bases or base analogs linkedtogether to form a polymer, including conventional RNA, DNA, mixedRNA-DNA, and analogs thereof.

The nitrogenous heterocyclic bases can be referred to as nucleobases.Nucleobases can be conventional DNA or RNA bases (A, G, C, T, U), baseanalogs, e.g., inosine, 5-nitroindazole L-nucleotides, and others (TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11.sup.thed., 1992; van Aerschott et al., 1995, Nucl. Acids Res. 23(21):4363-70), imidazole-4-carboxamide (Nair et al., 2001, NucleosidesNucleotides Nucl. Acids, 20(4-7):735-8), pyrimidine or purinederivatives, e.g., modified pyrimidine base6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (sometimes designated“P” base that binds A or G) and modified purine baseN6-methoxy-2,6-diaminopurine (sometimes designated “K” base that binds Cor T), hypoxanthine (Hill et al., 1998, Proc. Natl. Acad. Sci. USA95(8):4258-63, Lin and Brown, 1992, Nucl. Acids Res. 20(19):5149-52),2-amino-7-deaza-adenine (which pairs with C and T; Okamoto et al., 2002,Bioorg. Med. Chem. Lett. 12(1):97-9), N-4-methyl deoxygaunosine,4-ethyl-2′-deoxycytidine (Nguyen et al., 1998, Nucl. Acids Res.26(18):4249-58), 4,6-difluorobenzimidazole and 2,4-difluorobenzenenucleoside analogues (Kiopffer & Engels, 2005, Nucleosides NucleotidesNucl. Acids, 24(5-7) 651-4), pyrene-functionalized LNA nucleosideanalogues (Babu & Wengel, 2001, Chem. Commun. (Camb.) 20: 2114-5;Hrdlicka et al., 2005, J. Am. Chem. Soc. 127(38): 13293-9), deaza- oraza-modified purines and pyrimidines, pyrimidines with substituents atthe 5 or 6 position and purines with substituents at the 2, 6 or 8positions, 2-aminoadenine (nA), 2-thiouracil (sU),2-amino-6-methylaminopurine, O-6-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO-4-alkyl-pyrimidines (U.S. Pat. No. 5,378,825; PCT No. WO 93/13121;Gamper et al., 2004, Biochem. 43(31): 10224-36), and hydrophobicnucleobases that form duplex DNA without hydrogen bonding (Berger etal., 2000, Nucl. Acids Res. 28(15): 2911-4). Many derivatized andmodified nucleobases or analogues are commercially available (e.g., GlenResearch, Sterling, Va.).

A nucleobase unit attached to a sugar, can be referred to as anucleobase unit, or monomer. Sugar moieties of a nucleic acid can beribose, deoxyribose, or similar compounds, e.g., with 2′ methoxy or 2′halide substitutions. Nucleotides and nucleosides are examples ofnucleobase units.

The nucleobase units can be joined by a variety of linkages orconformations, including phosphodiester, phosphorothioate ormethylphosphonate linkages, peptide-nucleic acid linkages (PNA; Nielsenet al., 1994, Bioconj. Chem. 5(1): 3-7; PCT No. WO 95/32305), and alocked nucleic acid (LNA) conformation in which nucleotide monomers witha bicyclic furanose unit are locked in an RNA mimicking sugarconformation (Vester et al., 2004, Biochemistry 43(42):13233-41;Hakansson & Wengel, 2001, Bioorg. Med. Chem. Lett. 11 (7):935-8), orcombinations of such linkages in a nucleic acid strand. Nucleic acidsmay include one or more “abasic” residues, i.e., the backbone includesno nitrogenous base for one or more positions (U.S. Pat. No. 5,585,481).

A nucleic acid may include only conventional RNA or DNA sugars, basesand linkages, or may include both conventional components andsubstitutions (e.g., conventional RNA bases with 2′-O-methyl linkages,or a mixture of conventional bases and analogs). Inclusion of PNA,2′-methoxy or 2′-fluoro substituted RNA, or structures that affect theoverall charge, charge density, or steric associations of ahybridization complex, including oligomers that contain charged linkages(e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates)may affect the stability of duplexes formed by nucleic acids.

An oligomer may contain a “random polymer” sequence that refers to apopulation of oligomers that are substantially the same in overalllength and other characteristics, but in which at least a portion of theoligomer is synthesized by random incorporation of different bases for aspecified length, e.g., a random assortment of all four standard bases(A, T, G, and C) in a DNA oligomer, or a random assortment of a fewbases (U or T and G) in a defined portion of a larger oligomer. Theresulting oligomer is actually a population of oligomers whose finitenumber of members is determined by the length and number of bases makingup the random portion (e.g., 2⁶ oligomers in a population of oligomersthat contains a 6-nt random sequence synthesized by using 2 differentbases).

Complementarity of nucleic acids means that a nucleotide sequence in onestrand of nucleic acid, due to orientation of its nucleobase groups,hydrogen bonds to another sequence on an opposing nucleic acid strand.The complementary bases typically are, in DNA, A with T and C with G,and, in RNA, C with G, and U with A. Complementarity can be perfect(i.e., exact) or substantial/sufficient. Perfect complementarity betweentwo nucleic acids means that the two nucleic acids can form a duplex inwhich every base in the duplex is bonded to a complementary base byWatson-Crick pairing. “Substantial” or “sufficient” complementary meansthat a sequence in one strand is not completely and/or perfectlycomplementary to a sequence in an opposing strand, but that sufficientbonding occurs between bases on the two strands to form a stable hybridcomplex in set of hybridization conditions (e.g., salt concentration andtemperature). Such conditions can be predicted by using the sequencesand standard mathematical calculations to predict the Tm of hybridizedstrands, or by empirical determination of Tm by using routine methods.Tm refers to the temperature at which a population of hybridizationcomplexes formed between two nucleic acid strands are 50% denatured. Ata temperature below the Tm, formation of a hybridization complex isfavored, whereas at a temperature above the Tm, melting or separation ofthe strands in the hybridization complex is favored. Tm may be estimatedfor a nucleic acid having a known G+C content in an aqueous 1 M NaClsolution by using, e.g., Tm=81.5+0.41(% G+C), although other known Tmcomputations take into account nucleic acid structural characteristics.

“Separating” or “isolating” or “purifying” refers to removing one ormore components from a complex mixture, such as a sample. Preferably, aseparating, isolating or purifying step removes at least 70%, preferablyat least 90%, and more preferably at least 95% w/w of the target nucleicacids from other sample components. A separating, isolating or purifyingstep may optionally include additional washing steps to removenon-target sample components.

“Release” of a capture hybrid refers to separating one or morecomponents of a capture hybrid from each other, such as separating atarget nucleic acid from a capture probe, and/or a capture probe from animmobilized probe. Release of the target nucleic acid strand separatesthe target from other components of a capture hybrid and makes thetarget available for binding to a detection probe. Other components ofthe capture hybrid may remain bound, e.g., the capture probe strand tothe immobilized probe on a capture support, without affecting targetdetection.

A “label” refers to a molecular moiety that is detectable or produces adetectable response or signal directly or indirectly, e.g., bycatalyzing a reaction that produces a detectable signal. Labels includeluminescent moieties (such as fluorescent, bioluminescent, orchemiluminescent compounds), radioisotopes, members of specific bindingpairs (e.g., biotin and avidin), enzyme or enzyme substrate, reactivegroups, or chromophores, such as a dye or particle that results indetectable color.

A capture probe includes a first segment including atarget-complementary region of sequence and a second segment forattaching the capture probe, or a hybridization complex that includesthe capture probe, to an immobilized probe. The first segment can beconfigured to substantially complementary to a specific target nucleicacid sequence so that a first segment and a target nucleic acid canhybridize to form a stable duplex (i.e., having a detectable meltingpoint) under hybridizing conditions, such as described in the Examples.Alternatively, the first segment can be configured to nonspecificallybind to nucleic acid sequences in a sample under hybridizing conditions(see WO 2008/016988). The second segment includes a region of sequencethat is complementary to a sequence of an immobilized probe. Preferably,a chimeric capture probe includes a nucleic acid homopolymer (e.g.,poly-A or poly-T) that is covalently attached to thetarget-complementary region of the capture probe and that hybridizesunder appropriate conditions to a complementary homopolymer of theimmobilized probe (e.g., poly-T or poly-A, respectively) as previouslydescribed (U.S. Pat. No. 6,110,678 to Weisburg et al.). Capture probesmay further comprise a third segment that acts as a closing sequence toinactivate unbound target capture probes in a capture reaction. Thisthird segment can flank the first segment opposite the second segment(e.g., capture sequence:target hybridizing sequence:closing sequence) orit can flank the second segment opposite the first segment (e.g.,closing sequence:capture sequence:target hybridizing sequence). See WO2006/007567 and US 2009-0286249.

An immobilized probe includes a nucleic acid joined directly orindirectly to a support. The nucleic acid is complementary to a nucleicacid in the capture probe, although may or may not be the same length(number of nucleobase units) as the in the capture probe. The nucleicacid in the immobilized probe preferably contains at least sixcontiguous nucleobase units and can contain for example 10-45 or 10-40or 10-30 or 10-25 or 15-25, inclusively, L-nucleobase units. The nucleicacid is preferably a homopolymer, and more preferably a homopolymer ofadenine or thymine. A preferred form of immobilized probe is or includesa homopolymer of 14 thymine residues for use in combination with acapture probe including a second segment with a homopolymer of adenineresidues. The nucleic acid moiety of an immobilized probe is typicallyprovided in single-stranded form, or if not, is denatured tosingle-stranded form before or during use.

Any of a variety of materials may be used as a support for theimmobilized probes, e.g., matrices or particles made of nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene, and magnetically attractable materials. Monodispersemagnetic spheres are a preferred support because they are relativelyuniform in size and readily retrieved from solution by applying amagnetic force to the reaction container, preferably in an automatedsystem. An immobilized probe may be linked directly to the capturesupport, e.g., by using any of a variety of covalent linkages,chelation, or ionic interaction, or may be linked indirectly via one ormore linkers joined to the support. The linker can include one or morenucleotides not intended to hybridize to the capture probe but to act asa spacer between the nucleic acid of the immobilized probe and itssupport.

A “detection probe” is a nucleic acid or other molecule that bindsspecifically to a target sequence and which binding results, directly orindirectly, in a detectable signal to indicate the presence of thetarget sequence. A detection probe need not be labeled to produce adetectable signal, e.g., an electrical impulse resulting from bindingthe probe to its target sequence may be the detectable signal. A“labeled probe” is a probe that contains or is linked, directly orindirectly, to a label (e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, 2nd ed., Chapt. 10; U.S. Pat. No. 6,361,945, Beckeret al.; U.S. Pat. No. 5,658,737, Nelson et al.; U.S. Pat. No. 5,656,207,Woodhead et al.; U.S. Pat. No. 5,547,842, Hogan et al.; U.S. Pat. No.5,283,174, Arnold et al.; U.S. Pat. No. 4,581,333, Kourilsky et al.;U.S. Pat. No. 5,731,148, Becker et al.). For example, detection probesmay include a non-nucleotide linker and a chemiluminescent labelattached to the linker (U.S. Pat. Nos. 5,185,439, 5,585,481 and5,639,604, Arnold et al.). Examples of detection probes includeoligonucleotides of about 5 to 50 nucleotides in length having anattached label that is detected in a homogeneous reaction, e.g., onethat uses differential hydrolysis of a label on a bound or unboundprobe.

Detection probes can have a nucleotide sequence that is of the same oropposite sense as a target sequence depending on the format of theassay. Detection probes can hybridize to the same or different segmentof a target sequence as a capture probe. Some detection probes have anattached chemiluminescent marker, e.g., an acridinium ester (AE)compound (U.S. Pat. Nos. 5,185,439, 5,639,604, 5,585,481, and5,656,744). In some detection probes, an acridinium ester label isattached to a central region of the probe near a region of A and T basepairs by using a non-nucleotide linker (U.S. Pat. Nos. 5,585,481 and5,656,744, Arnold, et al.) which restricts the amines of the nucleotidebases on both sides of the AE and provides a site for intercalation.Alternatively, an AE label may be attached to the 3′ or 5′ terminus ofthe detection probe which is used in conjunction with a second oligomerthat hybridizes adjacent to the detection probe on the target nucleicacid to restrict the effects of nearby amine contributed by the targetnucleic acid. In some detection probes, an AE label at or near the siteof a mismatch with a related non-target polynucleotide sequence, topermit discrimination between the related sequence and the targetsequence that may differ by only one nucleotide because the area of theduplex around the mismatch site is sufficiently destabilized to renderthe AE on the probe hybridized to the related non-target sequencesusceptible to hydrolysis degradation. HIV-1 and HCV may be detectedusing a modified form of the commercial PROCLEIX® ULTRIO HIV-1/HCV/HBVAssay from Gen-Probe. The modification involves replacing the D-polyAand D-polyT sequences in capture and immobilized probes with L-poly Aand L-poly-T, respectively.

“Hybridization condition” refers to the cumulative environment in whichone nucleic acid strand bonds to a second nucleic acid strand bycomplementary strand interactions and hydrogen bonding to produce ahybridization complex. Such conditions include the chemical componentsand their concentrations (e.g., salts, chelating agents, formamide) ofan aqueous or organic solution containing the nucleic acids, and thetemperature of the mixture. Other factors, such as the length ofincubation time or reaction chamber dimensions may contribute to theenvironment (e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2.sup.nd ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).

Specific binding of a target capture oligomer to a target nucleic ortarget nucleic acids means binding between a single defined sequence inthe first segment of a target capture oligomer and an exactly orsubstantially complementary segment on target nucleic acid(s) to form astable duplex. Such binding is detectably stronger (higher signal ormelting temperature) than binding to other nucleic acids in the samplelacking a segment exactly or substantially complementary to the singledefined target capture oligomer sequence. Non-specific binding of atarget capture oligomer to target nucleic acids means that the targetcapture oligomer can bind to a population of target sequences that donot share a segment having exact or substantial complementarity to asingle defined target capture oligomer sequence. Such can be achieved byfor example using a randomized sequence in the first segment of thecapture probe.

Lack of binding between nucleic acids can be manifested by bindingindistinguishable from nonspecific binding occurring between a randomlyselected pair of nucleic acids lacking substantial complementarity butof the same lengths as the nucleic acids in question.

“Release” of a capture hybrid refers to separating one or morecomponents of a capture hybrid from each other, such as separating atarget nucleic acid from a capture probe, and/or a target captureoligomer from an immobilized probe. Release of the target nucleic acidstrand separates the target from other components of a capture hybridand makes the target available for binding to a detection probe. Othercomponents of the capture hybrid may remain bound, e.g., the targetcapture oligomer strand to the immobilized probe on a capture support,without affecting target detection.

“Sensitivity” is the proportion of true positives correctly identifiedas such (e.g. the percentage of infected patients correctly identifiedas having the infection). Specificity measures the proportion of truenegatives which are correctly identified (e.g. the percentage ofuninfected patients who are correctly identified as not having theinfection.)

Reference to a range of values also includes integers within the rangeand sub-ranges defined by integers in the range. Reference to anynumerical value or range of numerical values should be understand asencompassing any such variation as is inherent in measuring that valueother typical conditions of use.

DETAILED DESCRIPTION

I. General

The invention provides a lysis reagent for lysing red blood cells,thereby releasing RNA or other target in a form suitable for analysis.The lysis reagent includes at least ammonium chloride and an anionicdetergent, and may include an anti-coagulant. The reagent serves to lysered blood cells, protect a released target from degradation in thelysate, and is compatible with subsequent steps for analysis of thetarget, such as target capture, amplification, detection, and/orsequencing. Preferably, the lysis reagent preferentially lyses red bloodcells in a sample (e.g., whole blood) relative to white blood cells orother cells present to reduce contamination from lysates of other cellstypes and produce a homogeneous sample. The lysis reagent isparticularly amenable for analysis of RNA from pathogens infecting redblood cells, including parasitic organisms such as Babesia andPlasmodium species.

The invention results in part from identifying deficiencies with variousknown lysis agents for preparing and analyzing pathogen-derived RNA fromred blood cells. Known lysis agents were found to be incompatible withreagents and methods for analyzing pathogen-derived RNA, causing cellclumping, the appearance of precipitate, and the loss of magnetic beadswhen lysed samples were added to capture reagents. By contrast, thepresent lysis reagent was compatible with these methods, allowing forthe preferential lysis of red blood cells in whole blood samples and thesensitive detection of the released pathogen-derived RNA followingtarget capture and transcription mediated amplification. The presentlysis reagent also inhibited degradation of the pathogen-derived RNA bynucleases and proteases following lysis and demonstrated reproducibilitybetween samples.

II. Lysis Reagents

The present lysis reagent includes at least ammonium chloride and ananionic detergent, and preferably an anti-coagulant. Ammonium chloride(ACL) acts as a lysing agent. In whole blood, ammonium chloridepreferentially lyses red blood cells over white blood cells, thusreducing contamination of the sample and interference with steps of thedetection assay. Exemplary concentration ranges for ammonium chlorideinclude 100-1000 mM, 100-800 mM, 100-500 mM, 150-300 mM, 200-300 mM,240-260 mM, or 250 mM.

The anionic detergent can act as both a lysing agent and as an inhibitorof target degradation following the lysis of red blood cells. Theanionic detergent is particularly useful for inhibiting the degradationof nucleic acids. Exemplary anionic detergents include lithium laurylsulfate (LLS) or sodium dodecyl sulfate (SDS). LLS is preferred. By wayof example, a concentration range of 147 mM to 550 mM for LLS are 4-15%(w/v), 5-8% (w/v), or about 5% (w/v) of a stock LLS solution.

The anti-coagulant, if present, is used at a concentration sufficient toinhibit clotting of the sample (e.g., whole blood or red blood cells).By inhibiting clotting, the anti-coagulant eliminates the need tocentrifuge samples during the method to isolate red blood cells.Exemplary anti-coagulants include EDTA, heparin, or citrate. EDTA ispreferred. Exemplary concentrations of EDTA include 0.01-10 mM, 0.05-1.0mM, 0.05-0.50 mM, 0.075-0.125 mM, or 0.1 mM.

The lysis reagent can also include a buffer. Sodium bicarbonate is oneexample of a suitable buffer. Others suitable buffers are ACES, PIPES,MPSO, imidazole, Tris, BES, MOPS, HEPES, TES, MOBS, DIPSO, TAPSO,triethanolamine, pyrophosphate, HEPPSO, and POPSO. Sodium bicarbonate orother buffer can be present in the reagent at a concentration of, forexample 5-30 mM, 10-20 mM, 12-16 mM or 14 mM. The pH of the buffer usedin the reagent can be, for example, 7-8, or 7.2-7.6.

A preferred lysis reagent includes ammonium chloride, LLS, EDTA andsodium bicarbonate in a powder form or in a solvent, such as water, atany of the concentrations indicated above. Preferably ammonium chlorideis at a concentration of 100-500 mM or 250 mM, LLS at a concentration of4-15% or 5% (w/v), EDTA at a concentration of 0.01-10 mM or 0.1 mM, andsodium bicarbonate at a concentration of 12-16 mM or 14 mM, with a pH of7.2-7.6. Optionally, the lysis reagent consists essentially of ammoniumchloride, LLS, EDTA, sodium bicarbonate and water.

The lysis reagent can be provided as a kit also including probe and orprimers for performing an assay on a target to be isolated from redblood cells, including any of the targets described below. Such a kitcan include instructions for using the lysis reagent and/or performingan assay on a target isolated from red blood cells.

III. Use of Lysis Reagents

Red blood cells can be obtained from any available source, such as wholeblood or any fraction thereof that includes red blood cells, such aspelleted red blood cells. Whole blood can be human whole blood,non-human whole blood, or a combination thereof.

The lysis reagent can be admixed with red blood cells for a timesufficient to induce cell lysis and cause release of molecules ofdesired target(s) from cells. Exemplary times for maintaining red bloodcells admixed with lysis reagent include 1-30 minutes, 2-15 minutes,3-10 minutes, 4-6 minutes, or 5 minutes. Preferably, the time is no morethan 30, 15, 10 or 5 minutes. Preferably the mixture lacks visibleparticles after lysis.

The temperature of incubation of the lysis reagent with red blood cellscan vary. The temperature is preferably chosen to maximize extent andrate of lysis and preference for red blood cells over white blood cellsor other cells in the sample and to minimize degradation of target(s) orprevent inhibition of subsequent processing. Exemplary temperatureranges include 0-50° C., 5-45° C., 10-40° C., 15-37° C., 20-30° C.,22-27° C., or 25° C. Ambient temperature is suitable. Lysis of red bloodcells should release a sufficient amount of target molecules to bedetectable by the methods described herein. Preferably lysis results inat least 50%, 60%, 70%, 80%, 90%, or 100% lysis of red blood cells in asample being lysed.

The ratio at which whole blood is combined with lysis reagent can affectthe extent and rate of cell lysis and protection of target moleculesfrom degradation after release from lysed cells. Exemplary ratios inwhich whole blood is admixed with the lysis reagent include ratios of1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or in a range of ratios between 1:1 and1:10 (v/v; whole blood:reagent). A preferred ratio is whole bloodadmixed with the lysis reagent at a ratio of about 1:3 (v/v). When thesample comprises red blood cells isolated from whole blood, such aspelleted red blood cells, the red blood cells can be admixed with thelysis reagent at exemplary ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, orin a range of ratios between 1:1 and 1:10 (v/v; red bloodcells:reagent).

IV. Targets

Targets released from red blood cells by the present lysis reagent caninclude endogenous or pathogenic nucleic acids (e.g., DNA or RNA), wholeparticles, proteins from pathogenic viruses or organisms, and/orantibodies, lipids and carbohydrates. Pathogen-derived RNA targets arepreferred. Various types of RNA can be detected. The RNA can beribosomal RNA (rRNA), messenger RNA (mRNA), or heterogeneous nuclear RNA(hnRNA). A preferred target for pathogens is ribosomal RNA, particularly18S rRNA, 5S rRNA, 5.8S rRNA, or 28S rRNA.

Exemplary pathogenic organisms include parasites from the genus Babesia,Plasmodium, Trypanosoma, Leishmania, Anaplasma, or Toxoplasma. Organismsof the genus Babesia that cause disease in humans can be Babesiamicroti, Babesia divergens, or Babesia duncani. Organisms of the genusPlasmodium can be Plasmodium falciparum, Plasmodium malariae, Plasmodiumovale, Plasmodium vivax, or Plasmodium knowlesi.

V. Assays

Target molecules released from lysis of red blood cells are subject toanalysis. Target molecules may or may not be separated from the lysisreagent (by centrifugation or otherwise) before analysis. Omission of aseparation step can facilitate efficient work flow in performing theassay. The type of assay depends on the target.

A. Nucleic Acids

Analysis of nucleic acid targets often involves steps of capture,amplification and detection. Alternatively, amplification and detectionmethods can be performed without prior target capture. Preferablyamplification, and detection and target capture (if performed) occurwithout separation of target molecules from the lysis reagent. Thus, theentire process can be performed in a single vessel.

1. Target Capture Assay

An exemplary target capture assay can be performed as follows using oneor more capture probes, an immobilized probe, a sample, and a suitablemedium to permit hybridization of the target capture oligomer to thetarget nucleic acid and of target capture oligomer to the immobilizedprobe. The target sample can be heated (e.g., from 65° C. to 95° C.)before performing the assay to denature any nucleic acids indouble-stranded form. The components can be mixed in any order. Forexample the target capture oligomer can be added to the sample andhybridized with the target nucleic acid in the sample before adding theimmobilized probe. However, for an automated assay, it is preferable tominimize the number of adding steps by supplying the target captureoligomer and immobilized probe at the same or substantially the sametime. In this case, the order of hybridization can be controlled byperforming a first hybridization under conditions in which a duplex canform between the target capture oligomer and the target nucleic acid butwhich exceeds the melting temperature of the duplex that would formbetween first and second stem segments of the capture probe and betweenthe target capture oligomer and immobilized probe, and then performing asecond hybridization under conditions of reduced stringency, preferablybelow the melting temperature of the duplexes formed between the firstand second stem segments and between the target capture oligomer and theimmobilized probe. Stringency can be reduced by lowering the temperatureof the assay mix. At the higher temperature, the target binding siteduplexes with the target nucleic acid. At the lower temperature, thefirst and second stem segments of capture probes not bound to the targetnucleic acid duplex with one another and the first stem segment ofcapture probes bound to the target nucleic acid duplexes with theimmobilized probe. For example, the higher stringency hybridization canbe performed at or around 60° C. and the lower stringency hybridizationby allowing cooling to room temperature or 25° C. Stringency can also bereduced by reducing salt concentration or adding or increasingconcentration of a chaotropic solvent. In some methods, all steps (withthe possible exception of an initial denaturation step at highertemperature to denature double stranded target) can be performedisothermally.

Following formation of the target nucleic acid:capture probe,immobilized probe hybrid (the capture hybrid complex) is separated awayfrom other sample components by physically separating the capturesupport using any of a variety of known methods, e.g., centrifugation,filtration, or magnetic attraction of a magnetic capture support. Theseparation is preferably performed at a temperature below the meltingtemperature of stem-loop structures formed by target capture oligomersso that empty target capture oligomers have no opportunity to denatureand thus bind to the capture probe. In some methods, the separation isperformed at a temperature less than but within 10° C. of the meltingtemperature of the stem-loop structure (e.g., at 60° C.) to maintainstringency of hybridization conditions and consequent ability todistinguished matched and unmatched target nucleic acids.

To further facilitate isolation of the target nucleic acid from othersample components that adhere non-specifically to any portion of thecapture hybrid, the capture hybrid may be washed one or more times todilute and remove other sample components. Washing may be accomplishedby dissociating the capture hybrid into its individual components in anappropriate aqueous solution (e.g., a solution containing Tris and EDTA.See e.g., U.S. Pat. No. 6,110,678) and appropriate conditions (e.g.,temperature above the T_(m) of the components) and then readjusting theconditions to permit reformation of the capture hybrid. However, forease of handling and minimization of steps, washing preferably rinsesthe intact capture hybrid attached to the capture support in a solutionby using conditions that maintain the capture hybrid. Preferably,capture of the target nucleic acid with washing if performed, isolatesat least 70%, preferably at least 90%, and more preferably about 95% ofthe target nucleic acids away from other sample components. Isolatednucleic acids can be used for a number of downstream processes, such asnucleic acid amplification.

A target capture assay may also be performed as part of a real-time,biphasic, target capture and amplification method. In such a method, 500μL of sample and 400 μL of target capture reagent (TCR) are added toreaction tubes. The TCR contains magnetic particles, components to lyseorganisms present in the sample, capture oligos, a T7 initiationpromoter, and an internal calibrator. Fluid in the reaction tubes ismixed for a specific time and speed to ensure the mixture ishomogeneous. Reaction tubes are then transferred to a transitionincubator at 43.7° C. to preheat the fluid in the reaction tubes.Reaction tubes are then transferred to an anneal incubator set at 64° C.During incubation at 64° C., any organisms present in the sample thatwere not previously disrupted by the lysis reagent are disrupted,causing release of the target. Reaction tubes are then moved to atransition incubator to start a cool down process, and are furthercooled in a chiller ramp (17° C. to 19° C.), leading to binding of theT7 initiation promoter and capture of both the target and the internalcalibrator to the magnetic particles via the capture oligos. Thereaction tubes are moved to a magnetic parking station where they aresubjected to magnets which pull the magnetic particles to the sides ofthe tubes prior to entering a wash station. In the wash station,potential interfering substances are removed from the reaction bywashing the magnetic particles.

2. Amplification

A nucleic acid target can be amplified using methods such astranscription mediated amplification (TMA), polymerase chain reaction(PCR), Nucleic Acid Sequence-Based Amplification, ligase chain reactionor other amplification methods. Detection of the amplified target RNAproducts can be performed during amplification (real-time) or followingamplification (end-point).

i. Transcription Mediated Amplification

TMA has been previously described (e.g., U.S. Pat. Nos. 5,399,491,5,554,516, 5,824,518 and 7,833,716; and also e.g., F. Gonzales and S.McDonough. Applications of Transcription-Mediated Amplification toQuantification of Gene Sequences. Gene Amplification. 1998 Ed. FrancoisFerre, Birkhauser, Boston. PP. 189-204). In TMA, a target nucleic acidthat contains the sequence to be amplified is provided as singlestranded nucleic acid (e.g., ssRNA or ssDNA). Any conventional method ofconverting a double stranded nucleic acid (e.g., dsDNA) to asingle-stranded nucleic acid may be used. A promoter primer bindsspecifically to the target nucleic acid at its target sequence and areverse transcriptase (RT) extends the 3′ end of the promoter primerusing the target strand as a template to create a cDNA copy, resultingin a RNA:cDNA duplex. RNase activity (e.g., RNase H of RT enzyme)digests the RNA of the RNA:cDNA duplex and a second primer bindsspecifically to its target sequence in the cDNA, downstream from thepromoter-primer end. Then RT synthesizes a new DNA strand by extendingthe 3′ end of the second primer using the cDNA as a template to create adsDNA that contains a functional promoter sequence. RNA polymerasespecific for the functional promoter initiates transcription to produceabout 100 to 1000 RNA transcripts (amplified copies or amplicons)complementary to the initial target strand. The second primer bindsspecifically to its target sequence in each amplicon and RT creates acDNA from the amplicon RNA template to produce a RNA:cDNA duplex. RNasedigests the amplicon RNA from the RNA:cDNA duplex and thetarget-specific sequence of the promoter primer binds to itscomplementary sequence in the newly synthesized DNA and RT extends the3′ end of the promoter primer as well as the 3′ end of the cDNA tocreate a dsDNA that contains a functional promoter to which the RNApolymerase binds and transcribes additional amplicons that arecomplementary to the target strand. Autocatalytic cycles that use thesesteps repeatedly during the reaction produce about a billion-foldamplification of the initial target sequence. Optionally, amplicons maybe detected during amplification (real-time detection) or at an endpoint of the reaction (end-point detection) by using a probe that bindsspecifically to a sequence contained in the amplicons. Detection of asignal resulting from the bound probes indicates the presence of thetarget nucleic acid in the sample.

TMA may also be performed as part of a real-time, biphasic, targetcapture and amplification method. In such a method, TMA can be performedby adding amplification reagent (50 μL/test) to reaction tubescontaining captured target molecules and mixing in an amplification loadstation. The amplification reagent contains oligos and componentsnecessary to build nucleic acids. The reaction tubes are moved to atransition incubator at 43.7° C. to increase the temperature of theliquid in the reaction tubes, which are then moved back to theamplification load station where enzyme (25 μL/test) is added. Reactiontubes are moved to the amplification incubator set at 42.7° C. andremain in the incubator for five minutes, during which the first roundsof amplification are initiated. Reaction tubes are moved back to theamplification load station where promoter reagent (25 μL/test) is added.Reaction tubes are moved back to the amplification incubator for furtherrounds of target amplification. The promoter reagent contains oligos andtorches. The torches are complementary to the target or internalcalibrator and fluoresce when bound, generating signal in real-time. Thesignals for the target and internal calibrator preferably have differentwavelengths and can be distinguished.

ii. Polymerase Chain Reaction

Alternatively, PCR amplification (e.g., reverse transcriptase orreal-time PCR) can be used for amplification. PCR can be performed withor without prior release of the target nucleic acid from the capturecomplex. The PCR reaction can be performed in the same vessel (e.g., amicrofuge tube) as the capture step. The PCR reaction involvesthermocycling between a high temperature of about 95° C. (e.g., 90-99°C.) for dissociation and a low temperature of about 60° C. e.g., 40-75,or 50-70 or 55-64° C.) for annealing. Typically, the number of completethermocycles is at least 10, 20, 30 or 40. PCR amplification isperformed using one or more primer pairs. A primer pair used for PCRamplification includes two primers complementary to opposite strands ofa target nucleic acid flanking the region desired to be sequenced. Forsequencing most of a viral genome (e.g., more than 50, 75 or 99%), theprimers are preferably located close to the ends of the viral genome.For amplification of related molecules (e.g., mutant forms of the samevirus present in a patient sample), the primers are preferablycomplementary to conserved regions of the target nucleic acid likely tobe present in most members of the population. PCR amplification isdescribed in PCR Technology: Principles and Applications for DNAAmplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCRProtocols: A Guide to Methods and Applications (eds. Innis, et al.,Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic AcidsRes. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17(1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat.No. 4,683,202.

3. Detection

Detection of a nucleic acid target can be performed following captureand either during (real-time) or following (end-point) amplification byusing any known method. The amplification product of RNA is often in theform of DNA resulting from RT-PCR or RNA copies resulting from TMA.Amplified nucleic acids may be detected in solution phase or byconcentrating them in or on a matrix and detecting labels associatedwith them (e.g., an intercalating agent such as ethidium bromide). Somedetection methods use probes complementary to a sequence in theamplified product and detect the presence of the probe:product complex,or use a complex of probes to amplify the signal detected from amplifiedproducts (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503 and 5,849,481).Other detection methods use a probe in which signal production is linkedto the presence of the target sequence because a change in signalresults only when the labeled probe binds to amplified product, such asin a molecular beacon, molecular torch, or hybridization switch probe(e.g., U.S. Pat. Nos. 5,118,801, 5,210,015, 5,312,728, 5,538,848,5,541,308, 5,656,207, 5,658,737, 5,925,517, 6,150,097, 6,361,945,6,534,274, 6,835,542, and 6,849,412; and U.S. Pub. No. 2006/0194240 A1).Such probes typically use a label (e.g., fluorophore) attached to oneend of the probe and an interacting compound (e.g., quencher) attachedto another location of the probe to inhibit signal production from thelabel when the probe is in one conformation (“closed”) that indicates itis not hybridized to amplified product, but a detectable signal isproduced when the probe is hybridized to the amplified product whichchanges its conformation (to “open”). Detection of a signal fromdirectly or indirectly labeled probes that specifically associate withthe amplified product indicates the presence of the target nucleic acidthat was amplified.

4. Sequencing

Following amplification, a target nucleic acid as well as or instead ofundergoing qualitative or quantitative detection can be sequenced.Purification if desired can be performed on a silica column (e.g., aQiagen gravity flow column). The target nucleic acid binds to thecolumn, where it can be washed and then eluted. Alternatively,purification can be performed using a nucleic acid probe-basedpurification system (e.g., U.S. Pat. Nos. 6,110,678 or 8,034,554, US2013/0209992 or US 2009/0286249, or. WO 2012/037531 or WO 2013/116774).The amplified target DNA can also be adapted for some sequencing formatsby attachment of an adapter. The amplified DNA can be tailed byKlenow-mediated addition of nucleotides (usually a homopolymer) followedby annealing to an oligonucleotide complementary to the added tail, andligation. Depending on the sequencing platform used, special adaptorsare ligated to the template before sequencing. For example, a SMRT belladapter is ligated to the sample template for sequencing with a PacificBiosciences' PacBio RS sequencer (see, e.g., Travers et al. Nucl. AcidsRes. (2010) 38 (15): e159).

The amplified target nucleic acid is suitable for sequence analysis by avariety of techniques. The capture of target nucleic acid can be coupledto several different formats of so-called next generation and thirdgeneration sequencing methods. Such methods can sequence millions oftarget templates in parallel. Such methods are particularly useful whenthe target nucleic acid is a heterogeneous mixture of variants. Amongthe many advantages, sequencing variants in parallel provides a profileof drug resistant mutations in the sample, even drug mutations presentin relatively minor proportions within the sample.

Some next generation sequence methods amplify by emulsion PCR. A targetnucleic acid immobilized to beads via a target capture oligomer providesa suitable starting material for emulsion PCR. The beads are mixed withPCR reagents and emulsion oil to create individual micro reactorscontaining single beads (Margulies et al., Nature 437, 376-80 (2005)).The emulsion is then broken and the individual beads with amplified DNAare sequenced. The sequencing can be pyrosequencing performed forexample using a Roche 454 GS FLX sequencer (454 Life Sciences, Branford,Conn. 06405). Alternatively, sequencing can be ligation/detectionperformed for example using an ABI SOLiD Sequencing System (LifeTechnologies, Carlsbad, Calif. 92008). In another variation, targetnucleic acids are eluted from beads having target capture oligomers andare immobilized in different locations on an array (e.g., the HiScanSQ(Illumina, San Diego, Calif. 92121)). The target nucleic acids areamplified by bridge amplification and sequenced by template directedincorporation of labeled nucleotides, in an array format (Illumina). Inanother approach, target nucleic acids are eluted from the targetcapture oligomer and single molecules are analyzed by detecting inreal-time the incorporation nucleotides by a polymerase (single moleculereal time sequencing or SMRT sequencing). The nucleotides can be labelednucleotides that release a signal when incorporated (e.g., PacificBiosciences, Eid et al., Sciences 323 pp. 133-138 (2009) or unlabelednucleotides, wherein the system measures a chemical change onincorporation (e.g., Ion Torrent Personal Genome Machine (LifeTechnologies)).

Although captured target nucleic acids can be sequenced by anytechnique, third generation, next generation or massively parallelmethods offer considerable advantages over Sanger and Maxam Gilbertsequencing. Several groups have described an ultrahigh-throughput DNAsequencing procedure (see. e.g., Cheeseman, U.S. Pat. No. 5,302,509,Metzker et al., Nucleic Acids Res. 22: 4259 (1994)). The pyrosequencingapproach that employs four natural nucleotides (comprising a base ofadenine (A), cytosine (C), guanine (G), or thymine (T)) and severalother enzymes for sequencing DNA by synthesis is now widely used formutation detection (Ronaghi, Science 281, 363 (1998); Binladin et al.,PLoS ONE, issue 2, e197 (February 2007); Rehman et al., American Journalof Human Genetics, 86, 378 (March 2010); Lind et al., Next GenerationSequencing: The solution for high-resolution, unambiguous humanleukocyte antigen typing, Hum. Immunol. (2010), doi10.1016/jhumimm.2010.06.016 (in press); Shafer et al., J Infect Dis. 1;199(5):610 (2009)). In this approach, the detection is based on thepyrophosphate (PPi) released during the DNA polymerase reaction, thequantitative conversion of pyrophosphate to adenosine triphosphate (ATP)by sulfurylase, and the subsequent production of visible light byfirefly luciferase. More recent work performs DNA sequencing by asynthesis method mostly focused on a photocleavable chemical moiety thatis linked to a fluorescent dye to cap the 3′-OH group of deoxynucleosidetriphosphates (dNTPs) (Welch et al. Nucleosides and Nucleotides 18, 197(1999) & European Journal, 5:951-960 (1999); Xu et al., U.S. Pat. No.7,777,013; Williams et al., U.S. Pat. No. 7,645,596; Kao et al, U.S.Pat. No. 6,399,335; Nelson et al., U.S. Pat. Nos. 7,052,839 & 7,033,762;Kumar et al., U.S. Pat. No. 7,041,812; Sood et al, US Pat. App. No.2004-0152119; Eid et al., Science 323, 133 (2009)). Insequencing-by-synthesis methodology, DNA sequences are being deduced bymeasuring pyrophosphate release on testing DNA/polymerase complexes witheach deoxyribonucleotide triphosphate (dNTP) separately andsequentially. See Ronaghi et al., Science 281: 363 365 (1998); Hyman,Anal. Biochem. 174, 423 (1988); Harris, U.S. Pat. No. 7,767,400.

B. Other Targets

Antibodies, proteins, particles and other targets can be detected byformats such as immunoprecipitation, Western blotting, ELISA,radioimmunoassay, competitive and immunometric assays. See Harlow &Lane, Antibodies: A Laboratory Manual (CSHP NY, 1988); U.S. Pat. Nos.3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932;3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517;3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and4,098,876. Sandwich assays are a preferred format (see U.S. Pat. Nos.4,376,110, 4,486,530, 5,914,241, and 5,965,375).

Competitive assays can also be used. In some methods, target antigen ina sample competes with exogenously supplied labeled target antigen forbinding to an antibody detection reagent. The amount of labeled targetantigen bound to the antibody is inversely proportional to the amount oftarget antigen in the sample. The antibody can be immobilized tofacilitate separation of the bound complex from the sample prior todetection.

Lateral flow devices can also be used for detecting a target. Fluid isapplied to a test strip that has been treated with a sample in which atarget may be present. Labelled binding molecules pass through the stripand can be captured as they pass into a specific zone containing thesample with the target.

VI. Sensitivity

The present methods can provide a high sensitivity of detection of atarget from red blood cells. For pathogen-derived RNA targets,sensitivity can be expressed as a minimum number of pathogenic RNAcopies present in a volume of whole blood. The volume of whole blood canbe that contacted with lysis reagent directly, or can be that used toprepare a blood fraction, such as pelleted red cells, which are in turncontacted with the lysis reagent. Preferably the methods detect thepresence of pathogenic RNA in whole blood with a sensitivity of 2×10³copies of RNA equivalent to one parasite/1 mL of whole blood or better,2×10³ copies/5 mL of whole blood or better, 2×10³ copies/10 mL of wholeblood or better, 2×10³ copies/50 mL of whole blood or better, or 2×10³copies/100 mL of whole blood or better. In some methods, the range ofpathogenic RNA in lysed samples varies between 2×10³ to 2×10⁷ copies/1mL of a sample.

EXAMPLES Example 1. Analysis of Reagents for Cell Lysis andStabilization of Babesia RNA

The purpose of this example was to identify a lysis reagent that wouldeffectively lyse red blood cells in human whole blood, stabilizeBabesia-derived rRNA in the lysed sample, and inhibit the activity ofRNAses. To be compatible with Gen-Probe's Target Capture Technologyusing magnetic beads, the lysis reagent should preferably result in ahomogeneous lysate for efficient target capture.

The PAXGENE™ Blood RNA System (BD Biosciences) was first evaluated forBabesia sample preparation. The PAXGENE reagent contained in each tubecomprises the active compound tetradecyltrimethylammonium oxalate(TDTMAO), a quaternary ammonium salt known to lyse cell membranes andact as a stabilizing reagent.

The sample used for preparation was human whole blood spiked withBabesia-infected hamster blood at a dilution ranging from 1×10⁻¹ to1×10⁻⁶. The blood sample (1 mL) was added to 3 mL of lysis reagent froma PAXGENE tube at room temperature and allowed to rock for 5 minutes toinduce cell lysis. 500 μL of the lysed sample was then added to 500 μLof a Target Capture Reagent (TCR). Gen-Probe, PROCLEIX®, and APTIMA®TCRs were evaluated. Following addition of the lysed sample to the TCR,a white precipitate formed. Therefore, the PAXGENE system was unsuitablefor whole blood lysis and detection of Babesia using Gen-Probe's targetcapture reagents.

In a next experiment, a lysis reagent of 250 mM ammonium chloride (ACL),buffered with 14 mM sodium bicarbonate, was evaluated. Human whole bloodwas spiked with Babesia-infected hamster blood at a dilution rangingfrom 1×10⁻⁵ to 1×10⁻⁸. One mL of spiked whole blood was admixed with 3mL of buffered ACL solution for 5 minutes at 25° C. to induce red bloodcell lysis. Following the addition of 500 μL of the lysed sample to 500μL TCR, no precipitate was observed. Target capture was performed asgenerally described in U.S. Pat. No. 6,110,678. Babesia 18S rRNA wasdetected in each sample by transcription-mediated amplification (U.S.Pat. Nos. 5,399,491, 5,554,516, 5,824,518 and 7,833,716). Primers usedto amplify Babesia 18S rRNA in the samples were as follows:

TABLE 1 FUNCTION Sequence (5′-3′) non T7ACAGGGAGGTAGTGACAAG (SEQ ID NO: 1) Primer T7 PrimerAATTTAATACGACTCACTATAGGGAGACTGGAATTACC GCGGCTGCTGG (SEQ ID NO: 2)AE Probe ACCCUUCC CA GAGUAUCAAU (SEQ ID NO: 3) TCOGGAUUGGGUAAUUUGCGCGCCTTTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA (SEQ ID NO: 4)

As shown in FIG. 1, Babesia 18S rRNA was detected in the spiked humanwhole blood samples at a dilution as low as 1×10⁻⁸. However, a largevariability in performance was observed with regard to the age of theblood (3 to 5 days old). These results suggested that a stabilizationcomponent should be included if a buffered ACL solution were to be usedfor whole blood lysis and detection of Babesia.

Additional lysis reagents were evaluated for their compatibility withGen-Probe's Target Capture Technology. Lysis reagents evaluatedincluded: IC Buffer containing 10% lithium lauryl sulfate (LLS); RocheRBC lysis buffer; buffered ACL formulated with 4%tetradecyltrimethylammonium bromide (TDTMAB), 250 mM tartaric acid, and5% Tween 20 (HB+PTM1.1); Roche Stabilizing Reagent with 5% Tween-20,guanidinium thiocyanate, Triton X-100, and a reducing chemical; 0.2%saponin; 0.2% saponin formulated with 4% TDTMAB, 250 mM tartaric acid,and 10% Tween-20; 4% TDTMAB, 250 mM tartaric acid, and 10% Tween-20; andPAXGENE lysis solution. As shown in FIG. 2, only Roche RBC lysis bufferand IC buffer induced cell lysis in this experiment without theappearance of precipitate or loss of magnetic beads when added to TCR.

Further analysis of different lysis reagents was performed as shown inFIG. 3 for compatibility with PROCLEIX® and APTIMA® TCR solutions(Grifols SA, cat. no. 302573 and Hologic, Inc., cat. no.302178. A numberof reagents showed no or diminished activity in one or both TCRs(saponin with and without 5% Tween-20; PAXGENE lysis solution; PTM1.0,1.1, and 1.2). Some produced precipitate (PPT) when added to a TCR.Others were reasonable candidates in one or both TCRs, while TPM2.4 (250mM ACL, 14 mM sodium bicarbonate, and 5% LLS) was a good candidate inthe PROCLEIX® TCR and IC Buffer was a good candidate in both TCRs.

A study using the PTM 2.4 solution and IC buffer (10% LLS) was performedto compare the effectiveness of these lysis reagents for detectingBabesia. Human whole blood was spiked with Babesia-infected hamsterblood at dilutions ranging from 1×10⁻³ to 1×10⁶. Blood samples werelysed with a lysis reagent of buffered ACL and 5% LLS or a lysis reagentof IC buffer at a ratio of 1:3 (blood:reagent). Babesia 18S rRNA wasdetected in each sample using PROCLEIX® TCR and amplification by TMA asdescribed above. As shown in FIG. 4, Babesia 18S rRNA detection iscomparable when blood is lysed using PTM2.4 or IC Buffer. However, itwas observed in these experiments that, without an initialcentrifugation step to pellet red blood cells from whole blood, ICbuffer produced cellular debris following the lysis step that couldpotentially interfere with target capture and magnetic bead wash steps.Therefore, PTM2.4 (“buffered ACL and 5% LLS”) was selected for furtherevaluation.

Example 2. Evaluation of LLS in ACL Lysis Solution

The necessity of LLS in the buffered ACL solution was examined in thisexample. LLS was included in the buffered ACL solution at concentrationsof 0% (control), 1%, 2%, 3%, and 5%. Human whole blood was spiked withBabesia-infected hamster blood at dilutions ranging from 1×10⁻⁷ to1×10⁻⁹. Spiked whole blood samples were lysed with each of the bufferedACL/LLS solutions. Babesia 18S rRNA was detected in each sample usingPROCLEIX® TCR and amplification by TMA as described above. As shown inFIG. 5, a decrease in the concentration of LLS results in greatervariability of detection of the Babesia 18S rRNA. This is likely due toan insufficient amount of LLS present to stabilize the Babesia 18S rRNAtarget prior to amplification by TMA.

Example 3. Sensitivity of Detection of Babesia

The sensitivity of detecting Babesia in human whole blood using bufferedACL and 5% LLS was examined. Human whole blood was spiked withBabesia-infected hamster blood at dilutions of 1×10⁻⁴ to 1×10⁻¹¹. Bloodsamples were lysed with a lysis reagent of buffered ACL and 5% LLS.Babesia 18S rRNA was detected in each sample using PROCLEIX® TCR andamplification by TMA as described above. As shown in FIG. 6A, Babesia18S rRNA could be detected in human whole blood at dilutions as low as1×10⁻⁸ to 1×10⁻⁹ when using this lysis reagent and assay protocol.

The number of in vitro transcripts (IVTs) detectable by the method wasalso determined. The in vitro transcripts used in this experiment hadthe following sequence:

(SEQ ID NO: 5) GGGCGAAUUGGGUACCGGGCCCCCCCUCGAGGUCGACGCUUAGUAUAAGCUUUUAUACAGCGAAACUGCGAAUGGCUCAUUAAAACAGUUAUAGUUUAUUUGAUGUUCGUUUUACAUGGAUAACCGUGGUAAUUCUAGGGCUAAUACAUGCUCGAGGCGCGUUUUCGCGUGGCGUUUAUUAGACUUUAACCAACCCUUCGGGUAAUCGGUGAUUCAUAAUAAAUUAGCGAAUCGCAUGGCUUUGCCGGCGAUGUAUCAUUCAAGUUUCUGACCUAUCAGCUUUGGACGGUAGGGUAUUGGCCACCGGGGCGACGACGGGUGACGGGGAAUUGGGGUUCGAUUCCGGAGAGGGAGCCUGAGAAACGGCUACCACAUCUAAGGAAGGCAGCAGGCGCGCAAAUUACCCAAUCCUGACACAGGGAGGUAGUGACAAGAAAUAACAAUACAGGGCUUAAAGUCUUGUAAUUGGAAUGAUGGGAAUCUAAACCCUUCCCAGAGUAUCAAUUGGAGGGCAAGUCUGGUGCCAGCAGCCGCGGUAAUUCCAGCUCCAAUAGCGUAUAUUAAAGUUGUUGCAGUUAAGAAGCUCGUAGUUGAAUUUCUGCCUUGUCAUUAAUCUCGCUUCCGAGCGUUUUUUUAUUGACUUGGCAUCUUCUGGAUUUGGUGCCUUCGGGUACUAUUUUCCAGGAUUUACUUUGAGAAAACUAGAGUGUUUCAAACAGGCAUUCGCCUUGAAUACUACAGCAUGGAAUAAUGAAGUAGGACUUUGGUUCUAUUUUGUUGGUUAUUGAGCCAGAGUAAUGGUUAAUAGGAGCAGUUGGGGGCAUUCGUAUUUAACUGUCAGAGGUGAAAUUCUUAGAUUUGUUAAAGACGAACUACUGCGAAAGCAUUUGCCAAGGAUGUUUUCAUUAAUCAAGAACGAAAGUUAGGGGAUCGAAGACGAUCAGAUACCGUCGUAGUCCUAACCAUAAACUAUGCCGACUAGAGAUUGGAGGUCGUCAGUUUAAACGACUCCUUCAGCACCUUGAGAGAAAUCAAAGUCUUUGGGUUCUGGGGGGAGUAUGGUCGCAAGUCUGAAACUUAAAGGAAUUGACGGAAGGGCACCACCAGGCGUGGAGCCUGCGGCUUAAUUUGACUCAACACGGGAAACCUCACCAGGUCCAGACAUAGAGAGGAUUGACAGAUUGAUAGCUCUUUCUUGAU GAAUU

The analytical sensitivity of the assay was determined by using in vitrotranscripts (IVT) of the Babesia 18s rRNA target. 10, 100, 1000, or10,000 IVT copies of Babesia 18S rRNA were spiked into IC buffer. IVTwas detected using PROCLEIX® TCR and procedure to capture the IVT,followed by TMA amplification of the captured IVT, as described above.As shown in FIG. 6B, as few as 10 IVT copies per mL could be detected.

The number of Babesia parasites detectable by the assay was calculatedbased on these results. As indicated from a parasitemic smear ofBabesia-infected hamster blood, approximately 5.04×10⁶ out of 7.20×10⁶red blood cells per uL were infected (˜70% of RBCs). Thus, at a dilutionof 1×10⁻⁶ to 1×10⁻⁷, where Babesia 18S rRNA is detectable by the assayusing buffered ACL and 5% LLS, approximately 1-10 parasites weredetectable in a volume of 1 mL of spiked human whole blood sample. Asshown in FIG. 6A, the method can detect Babesia 18S rRNA at dilutions aslow as 1×10⁻⁹, indicating that the sensitivity of the method can be aslow as 1-10 parasites per 100 mL of sample, or lower, correlating to2×10³ to 2×10⁴ 18s rRNA copies per 100 mL of human whole blood.

Example 4. Determining the Optimal Ratio of Blood to Lysis Reagent

The purpose of this example was to determine the optimal ratio of wholeblood to lysis reagent for use in the method. Human whole blood wasspiked with Babesia-infected hamster blood at a dilution ranging from1×10⁻⁴ to 1×10⁻¹¹. The samples were then lysed with a lysis reagent ofbuffered ACL and 5% LLS at a ratio of 1:2 or 1:3 (blood:reagent).Babesia 18S rRNA was detected in each sample using PROCLEIX® TCR andamplification by TMA as described above. As shown in FIG. 7, a ratio of1:3 (blood:reagent) was superior for the detection of low concentrationsof Babesia in human whole blood, with sensitivity as low as 1×10⁻⁹. Bycontrast, the sensitivity of detection was reduced when using a ratio of1:2 (blood:reagent).

Example 5. Reproducibility of Babesia Detection Between Samples

The reproducibility of the Babesia detection assay was determined acrossmultiple samples of donor blood. In this experiment, human whole bloodwas collected from six different donor patients. Each sample was spikedwith Babesia-infected hamster blood at dilutions between 1×10⁻⁶ to1×10⁻¹². Samples were then lysed with a lysis reagent of buffered ACLand 5% LLS. Babesia 18S rRNA was detected in each sample using PROCLEIX®TCR and amplification by TMA as described above. As shown in FIG. 8, useof the lysis reagent with this protocol produced excellentreproducibility between different donor samples for the detection ofBabesia 18S rRNA at dilutions as low as 1×10⁻⁶ and 1×10⁻⁸.

Example 6. Stability of Babesia Parasites and Nucleic Acids

Experiments were conducted to determine the stability of Babesiaparasites in human whole blood over time. To determine parasitestability, human whole blood was spiked with Babesia-infected hamsterblood at dilutions ranging from 1×10⁻² to 1×10⁻⁹. Prior to analysis, onegroup of samples was stored at 25° C. for five days after spiking.Another group was analyzed the same day that the human whole blood wasspiked. Spiked whole blood samples were lysed using a lysis reagent ofbuffered ACL and 5% LLS. Babesia 18S rRNA was detected in each sampleusing PROCLEIX® TCR and amplification by TMA as described above. Asshown in FIG. 9, a significant loss of sensitivity was observed afterfive days of storage at 25° C., particularly at dilutions of 1×10⁻⁷ and1×10⁻⁸.

An additional study was performed to determine the stability of Babesia18S rRNA in samples following RBC lysis. Human whole blood was spikedwith Babesia-infected hamster blood at dilutions ranging between 1×10⁻⁶to 1×10⁻⁹. The samples were lysed using a lysis reagent of buffered ACLand 5% LLS. Lysed samples were stored at 4° C. for 0-4 days prior toanalysis. Babesia 18S rRNA was detected in each sample using PROCLEIX®TCR and amplification by TMA as described above. As shown in FIG. 10, aloss of sensitivity was observed following storage at 4° C.,particularly after three and four days. This loss in sensitivity wasreadily observable in samples having a dilution of 1×10⁻⁹.

Although the invention has been described in detail for purposes ofclarity of understanding, certain modifications may be practiced withinthe scope of the appended claims. All publications including accessionnumbers, websites and the like, and patent documents cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each were so individually denoted.To the extent difference version of a sequence, website or otherreference may be present at different times, the version associated withthe reference at the effective filing date is meant. The effectivefiling date means the earliest priority date at which the accessionnumber at issue is disclosed. Unless otherwise apparent from the contextany element, embodiment, step, feature or aspect of the invention can beperformed in combination with any other.

What is claimed is:
 1. A method for isolating a target nucleic acid fromred blood cells, the method comprising: (a) mixing a sample containingred blood cells with a reagent comprising ammonium chloride at aconcentration of 200-300 mM, lithium lauryl sulfate (LLS) at aconcentration of 4% to 15% (w/v), and an anti-coagulant; (b) incubatingthe mixture of step (a), wherein the red blood cells are lysed and atarget nucleic acid in the red blood cells is released; (c) adding atarget capture oligomer and an immobilized probe to the mixture, whereinthe target capture oligomer comprises (i) a target hybridizing sequencethat hybridizes with the target nucleic acid and (ii) a segment forattaching the target capture oligomer to the immobilized probe, therebyforming a capture hybrid complex comprising the target capture oligomerand the target nucleic acid; and (d) separating the capture hybridcomplex away from other components of the sample, thereby isolating thetarget nucleic acid from the red blood cells.
 2. The method of claim 1,wherein the target nucleic acid is a pathogen-derived target.
 3. Themethod of claim 1, wherein the target nucleic acid is RNA.
 4. The methodof claim 2, wherein the target nucleic acid is RNA.
 5. The method ofclaim 1, wherein the mixture further comprises a buffer.
 6. The methodof claim 5, wherein the buffer is sodium bicarbonate.
 7. The method ofclaim 6, wherein the concentration of sodium bicarbonate is 5-30 mM. 8.The method of claim 7, wherein the concentration of sodium bicarbonateis 10-20 mM.
 9. The method of claim 6, wherein the pH of the buffer is7-8.
 10. The method of claim 1, wherein the anti-coagulant is EDTA orcitrate.
 11. The method of claim 1, wherein the concentration ofammonium chloride in the reagent is 250 mM.
 12. The method of claim 1,wherein the concentration of LLS in the reagent is 5% to 8% (w/v). 13.The method of claim 12, wherein the concentration of LLS in the reagentis 5% (w/v).
 14. The method of claim 1, wherein the concentration ofammonium chloride is 250 mM, the concentration of LLS is 5% (w/v), theconcentration of sodium bicarbonate is 14 mM, and the pH is 7.2-7.6. 15.The method of claim 1, wherein the segment for attaching the targetcapture oligomer to the immobilized probe is a poly-A sequence.
 16. Themethod of claim 1, wherein the immobilized probe is attached to amagnetic support.
 17. The method of claim 1, wherein the capture hybridcomplex is separated away from other components of the sample usingmagnetic separation.
 18. A method for isolating a target nucleic acidfrom red blood cells, the method comprising: (a) mixing a samplecontaining red blood cells with a reagent comprising ammonium chlorideat a concentration of 100-500 mM, lithium lauryl sulfate (LLS) at aconcentration of 4% to 15% (w/v), sodium bicarbonate buffer at aconcentration of 5-30 mM, and an anti-coagulant; (b) incubating themixture of step (a), wherein the red blood cells are lysed and a targetnucleic acid in the red blood cells is released; (c) adding a targetcapture oligomer and an immobilized probe to the mixture, wherein thetarget capture oligomer comprises (i) a target hybridizing sequence thathybridizes with the target nucleic acid and (ii) a segment for attachingthe target capture oligomer to the immobilized probe, thereby forming acapture hybrid complex comprising the target capture oligomer and thetarget nucleic acid; and (d) separating the capture hybrid complex awayfrom other components of the sample, thereby isolating the targetnucleic acid from the red blood cells.
 19. The method of claim 18,wherein the concentration of LLS in the reagent is 5% to 8% (w/v). 20.The method of claim 18, wherein the capture hybrid complex is separatedaway from other components of the sample using magnetic separation. 21.A method for isolating a target nucleic acid from red blood cells, themethod comprising: (a) mixing a sample containing red blood cells with areagent comprising ammonium chloride at a concentration of 100-500 mM,lithium lauryl sulfate (LLS) at a concentration of 4% to 15% (w/v),sodium bicarbonate buffer at pH 7-8, and an anti-coagulant; (b)incubating the mixture of step (a), wherein the red blood cells arelysed and a target nucleic acid in the red blood cells is released; (c)adding a target capture oligomer and an immobilized probe to themixture, wherein the target capture oligomer comprises (i) a targethybridizing sequence that hybridizes with the target nucleic acid and(ii) a segment for attaching the target capture oligomer to theimmobilized probe, thereby forming a capture hybrid complex comprisingthe target capture oligomer and the target nucleic acid; and (d)separating the capture hybrid complex away from other components of thesample, thereby isolating the target nucleic acid from the red bloodcells.
 22. The method of claim 21, wherein the concentration of LLS inthe reagent is 5% to 8% (w/v).
 23. The method of claim 21, wherein thecapture hybrid complex is separated away from other components of thesample using magnetic separation.